17-KDA Brucella abortus antigen, recombinant polypeptides, nucleic acids coding for the same and use thereof in diagnostic and prophylactic methods and kits

ABSTRACT

The present invention relates to an isolated 17-kDa Brucclla antigen characterized by an amino acid sequence having at least 60% homology, preferably at least 70% homology, more preferably having at least 80% homology to the amino acid sequence as shown in SEQ ID No. 1 or 2, with said antigen being specifically recognized by sera from Brucella field infected individuals, more particulary an antigen characterized by the amino acid sequence as shown in SEQ ID No. 1 or 2. The invention also relates to recombinantly produced 17 kDa Brucella antigen, nucleic acids coding for the same and the use thereof in diagnostic and prophylactic methods and kits.

The present invention relates to an isolated and pure 17-kDa Brucellaabortus antigen, which can be used for the diagnosis of Brucellainfection in human and cattle. The invention also relates to nucleicacids coding for said antigen, as well as to diagnostic methods and kitsusing such nucleic acids for detecting Brucella infection. The inventionalso relates to recombinant polypeptides, a process for preparing thesame and their use in methods and kits for the diagnosis of Brucellainfection. The invention also relates to the possible use of saidisolated antigen or said recombinant polypeptides as an active principleof a vaccine composition against Brucella strains. The invention relatesalso to a vaccine composition comprising a recombinant Brucella strain,specifically deleted for the gene encoding said antigen.

Brucellosis is an infection due to a small intracellular gram-negativebacterium which is pathogenic for humans as well as for domesticanimals. This infection induces abortions in livestock animals leadingto severe economic losses. Within the genus Brucelia, six closelyrelated species have been described (Fekete et al., 1992; Verger et al.,1985; Verstraete & Winter, 1984), the most important of which are B.abortus and B. melitensis. Humans and ruminants (sheep, goats and cows)are predominantly infected by these two Brucelia strains. Serologicaltests currently used for diagnosis of brucellosis infection are based onthe detection of anti-lipopolysaccharide (LPS) antibodies (Alton et al.,1988). These tests do not permit the differentiation between vaccinatedand infected animals, and fail to reveal some infected animals which arepositive in an intradermic test. Moreover important cross-reactions withother gram-negative bacteria have been reported (Corbel et al., 1983;Perry & Bundle, 1990; Schoerner et al., 1990). Diagnostic tests withhigher specificity are based on the isolation of Brucella bacteria or onthe intradermic injection of a protein preparation from Brucellabacteria “Brucellergen” (Fensterbank, 1984), leading to a delayed typehypersensitivity reaction (DTH). However, classical bacteriology istime-consuming and “Brucellergen” preparations are not always easy toproduce free of LPS. The latter can cause seroconversion of animals uponDTH testing, precluding its serological distinction from infectedanimals. The identification of specific antigens for brucellosisdiagnosis is therefore a matter of great interest for the development ofa specific serological test.

For prophylactic vaccination against brucellosis, today two live vaccinestrains are being used succesfully. The B19 strain is mostly used incattle and the Rev. 1 strain in small ruminants. The H38 killed vaccinehas also been used. Although good protection is generally obtained withthese vaccines, the general drawback is the induction of an immuneresponse in the vaccinated animals, which precludes the distinctionbetween infected and vaccinated animals.

A Brucella abortus cytoplasmic preparation was described by Goldbaum etal. (1993) which showed several bands of different molecular weights.The two major components are those of 18- and 36-kDa. This cytoplasmicpreparation was obtained by immunoadsorption of a B. abortus cytoplasmicfraction with an IgG2b monoclonal antibody (BI24) produced by immunizingwith a LPS-free cytoplasmic antigenic fraction preparation (LPS-freeCYT) as antigen. Microsequencing of the identified cytoplasmicpreparation (including the 18- and 36-kDa bands) resulted in theelucidation of the sequence of only the following three trypticpeptides:

(i) SEQ ID NO: 6

(ii) SEQ ID NO: 7

(iii) SEQ ID NO: 8

Another cytoplasmic Brucella protein preparation with an apparentmolecular mass of 20 kDa has been described by Zygmunt et al. (1992).

Cloeckaert et al. (1990, 1991) describe the production of 26anti-Brucella OMP monoclonal antibodies to R and S Brucella cellsdirected against seven outer membrane protein components.

The aim of the present invention is to provide a Brucella antigen or apolynucleic acid encoding the same which is useful for diagnosing invitro Brucella infection (=brucellosis) in mammals (humans, ruminants).

A special aim of the invention is to provide a Brucella antigen or apolynucleic acid encoding the same which is useful for differentiatingbetween field infected and vaccinated individuals.

Another aim of the present invention is to provide purified and isolated17 kDa antigen of Brucelia, and more particularly purified and isolatedB. abortus 17 kDa antigen.

Another aim of the present invention is to provide amino acid andcorresponding nucleotide sequences of said Brucella 17 kDa antigen.

Another aim of the present invention is to provide antibodiesspecifically directed against said Brucella 17 kDa antigen.

Another aim of the present invention is to provide primers and probesderived from the nucleotide sequences encoding said Brucella 17 kDaantigen.

Another aim of the present invention is to provide diagnostic methods orkits for diagnosing Brucella infection, more particularly fordifferentiating between field infected and vaccinated individuals, basedon any of the above-mentioned polypeptides or polynucleic acids asactive compounds.

Another aim of the present invention is to provide vaccine compositionsproviding protective immunity towards brucellosis in mammals (humans,ruminants).

Another aim of the present invention is to provide a recombinantBrucella strain in which the gene encoding the Brucella 17 kDa antigenhas been deleted or inactivated.

The present invention relates more particularly to an isolated 17-kDaBrucella antigen characterized by an amino acid sequence having at least60% homology, preferably at least 70% homology, more preferably at least80% homology to the 158 residue amino acid sequence as shown in SEQ IDNO 2, or fragments of said antigen, consisting of at least 9 contiguousamino acids from said amino acid sequence.

The term “isolated” refers to a purity grade of at least 90%, preferably95% and more preferably of 98% of the antigen expressed in weight versuscontaminants, as determined by one or two dimensional SDS-PAGE. Saidpurity may be obtained by purification of the naturally occurringpolypeptide, or by de novo synthesis of the polypeptide, by chemicalmethods or by recombinant DNA technology, and subsequent purification.The term “isolated” thus implies that the antigen is in a differentstate and environment than the naturally occurring antigen.

The word “antigen” refers to a molecule which provokes an immuneresponse (also called “immunogen”), or which can be recognized by theimmune system (also called “antigen sensu strictu”). The immune responseor the immune recognition reaction can be of the cellular or humoraltype.

The term “17 kDa antigen” refers to an antigen having a molecular weightof approximately 17 kDa as determined by SDS-PAGE. Said determinedmolecular weight may vary according to strain to strain variations, oraccording to methodology variations. Preferably, the molecular weight ofthe antigen of the invention as determined by SDS-PAGE is between 14 and20 kDa, more preferably between 15 and 19 kDa, most preferably between16 and 18 kDa.

The terms “homologous” and “homology” are used in the current inventionas synonyms for “identical” and “identity”; this means that amino acidsequences which are e.g. said to be 55% homologous, show 55% identicalamino acids in the same position upon alignment of the sequences.

In an effort to identify a purified and isolated Brucella 17-kDaantigen, an expression library containing B. abortus chromosomal DNAinserts was screened with sera from field infected sheep (see Examplessection). The DNA insert from a positive recombinant phage was analyzedin detail. Sequence analysis revealed a new gene encoding a 17-kDaBrucella abortus protein (SEQ ID NO 1). The gene was recombinantlyexpressed, and the antigenicity of the recombinantly produced newprotein was analyzed in Western blotting, in ELISA using sera frominfected humans and ruminants (sheep, goats, cows), and in competitiveELISA with a panel of Brucella monoclonal antibodies, to evaluate itssignificance for serological diagnosis of Brucella infected individuals.

According to a preferred embodiment, the invention relates to apolypeptide or peptide comprising in its amino acid sequence part of theamino acid sequence as represented in SEQ ID NO 2, said part consistingof at least 9 contiguous amino acids of the amino acid sequencerepresented in SEQ ID NO 2.

In a very specific embodiment the invention relates to an isolated17-kDa Brucella abortus antigen characterized by the 158 residue aminoacid sequence as shown in SEQ ID NO 2, or a fragment thereof, saidfragment consisting of at least 9 contiguous amino acids of the aminoacid sequence represented in SEQ ID NO 2.

The fragments of the above-mentioned polypeptides are preferably morethan 8, more preferably more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 50, 55 or 60 amino acids residues long, said aminoacids being contiguous amino acids selected from the amino acidsequences of any of the polypeptides described above.

The 17-kDa Brucella abortus polypeptide of SEQ ID NO 2, and thecorresponding nucleic acid of SEQ ID NO 1, are new.

A preferred embodiment of the present invention relates to any of thepolypeptides or polypeptide fragments as described above, with saidpolypeptides or polypeptide fragments having at least one of thefollowing immunological (antigenic) properties:

being specifically recognized by sera from Brucella field infectedindividuals, and/or

being specifically recognized by the cellular immune response fromBrucella contacted individuals, and/or

being able to elicit a Brucella specific immune response uponvaccination of individuals prone to brucellosis disease.

Of the above-mentioned immunological properties, the specificrecognition by sera from field-infected individuals is the mostpreferential one.

An additional characteristic of some of the polypeptides of theinvention is the differential recognition by sera from field-infectedindividuals as opposed to sera from vaccinated individuals.

The wording “field infected individuals” as used in the currentinvention refers to individuals which have been infected by the Brucellapathogen, and in which the infection lead to brucellosis disease.

The wording “Brucella vaccinated individuals” as used in the currentinvention refers to those individuals which have been vaccinated againstbrucellosis, and in which the vaccination has lead to protection of theindividual against active disease and against vertical transmission ofthe pathogen.

The wording “Brucella contacted individuals” as used in the currrentinvention refers to individuals which have been in contact with theBrucella pathogen, said contact possibly leading to subsequent disease,or to subsequent protection.

The expression “specifically recognized by sera from Brucella fieldinfected individuals” refers to the fact that the 17 kDa Brucellaantigen of the present invention shows preferentially a positiveimmunological reaction with sera from Brucella field infectedindividuals, whereas it shows preferentially no raction with sera from“control” individuals (such as healthy individuals or individualsinfected by other pathogens). Thus, sera originating from Brucella fieldinfected individuals are likely to contain antibodies reacting with thisantigen, while sera originating from control individuals are likely notto contain antibodies recognizing the 17-kDa Brucella antigen of theinvention. This will be demonstrated in the Examples section.

Preferentially, at least 30%, more preferably at least 40%, mostpreferably at least 50% or even at least 60% of the sera from fieldinfected individuals react with the polypeptides of the invention, whileat most 10%, preferably at most 5, and most preferably at most 1% of thesera from control individuals react with the polypeptides of theinvention.

It should be understood that, since the above-mentioned reactivities areusually a result of optical density measurements, e.g. in a standardELISA assay, positive and negative values have to be considered againsta so called “cutoff value”, which is not an absolute value, but whichcan be calculated from the optical density values of the control sera(e.g. cutoff value=mean optical density of the control sera+2 (or 3)standard deviations). This is demonstrated further in the examplessection.

The term “individual” refers to an animal or human being liable to beinfected by Brucella (species). Brucellosis infects mammals, mostlyhumans and ruminants.

The expression “to elicit a Brucella specific immune response” refers tothe fact that said 17 kDa Brucella immunogen according to the inventionis able to give rise to the production of antibodies which are specificfor Brucella, or that the 17 kDa Brucella immunogen elicits a cellularimmune response specific for Brucella, in individuals liable to beinfected by Brucella (species).

The expresssion “specifically recognized by the cellular immuneresponse” throughout the current application refers to the recognitionof the polypeptides of the invention by the T-cell population of theBrucella contacted individuals, said recognition being detectable invitro e.g. by lymphoproliferation tests in presence of the polypeptidesof the invention, or in vivo e.g. by a delayed type hypersensitivityreaction upon subcutaneous injection of the polypeptides of theinvention in the individual.

As disclosed above, the present invention also relates to antigenscharacterized by an amino acid sequence showing a homology of at least60%, preferably at least 70%, and even more preferably at least 80% or90% to the amino acid sequence of the new Brucelia abortus protein asdepicted in SEQ ID NO 2. Said “related” antigens may also be referred toas “muteins” of the protein shown in SEQ ID NO 2. The term “muteins” maybe defined as proteins containing substitutions and/or deletions and/oradditions of one or several amino acids, provided that said muteins haveretained the antigenic/immunogenic properties of the Brucella proteinsof the invention, i.e.:

being specifically recognized by sera from Brucella field infectedindividuals, and/or

being specifically recognized by the cellular immune response fromBrucella contacted individuals, and/or

being able to elicit a Brucella specific immune response uponvaccination of individuals prone to brucellosis disease.

An overview of the amino acid substitutions which could form the basisof such muteins are given in Table 1.

Said “muteins” may be the result of strain to strain variations of theBrucella (species) 17 kDa antigen, or may be the result of modificationsintroduced in the original polypeptide sequences, said modificationsbringing about a desirable side effect to the polypeptide molecule (e.g.better physicochemical properties, more efficient purification, moreefficient coating characteristics, more stable etc . . . ).

The 17 kDa antigen of Brucella abortus, represented by SEQ ID NO 2, hashomologous counterparts in other Brucella species, like e.g. Brucellamelitensis, Brucella ovis, Brucella suis, etc. These homologous genesand proteins are also part of the present invention.

All polypeptides according to the present invention, including muteinsand homologues, will be further referred to as “Brucella 17 kDaantigen”.

It should also be evident that such muteins and homologues, althoughfalling within the above-given definitions, might have a molecularweight which is slightly different from 17 kDa as determined bySDS-PAGE.

Peptides according to the present embodiment of the invention can bereadily determined by the person skilled in the art by applying any ofthe techniques teached in the Examples and Description sections of thepresent invention or any other immunological and epitope mappingtechniques known in the art.

According to an even more preferred embodiment, the present inventionrelates to peptidic fragments of Brucella 17-kDa antigen being able todistinguish Brucella field infected from Brucella vaccinated individualsupon incubation with sera originating from those individuals.

Preferably the peptides of the invention are different from a peptidewith amino acid sequence SEQ ID NO: 8.

The words “polypeptide” and “peptide” are used interchangeablythroughout the specification and designate a linear series of aminoacids connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent amino acids. Polypeptides canbe in a variety of lengths, either in their natural (uncharged) forms orin forms which are salts, and either free of modifications such asglycosylation, side chain oxidation, or phosphorylation or containingthese modifications. It is well understood in the art that amino acidsequences contain acidic and basic groups, and that the particularionization state exhibited by the peptide is dependent on the pH of thesurrounding medium when the protein is in solution, or that of themedium from which it was obtained if the protein is in solid form. Alsoincluded in the definition are proteins modified by additionalsubstituents attached to the amino acids side chains, such as glycosylunits, lipids, or inorganic ions such as phosphates, as well asmodifications relating to chemical conversions of the chains, such asoxidation of sulfhydryl groups. Thus, “polypeptide” or its equivalentterms is intended to include the appropriate amino acid sequencereferenced, subject to those of the foregoing modifications which do notdestroy its functionality.

The polypeptides of the invention, and particularly the shorterpeptides, can be prepared by classical chemical synthesis.

The synthesis can be carried out in homogeneous solution or on solidphase.

For instance, the synthesis technique in homogeneous solution which canbe used is the one described by Houbenweyl in the book entitled “Methodeder organischen chemie” (Method of organic chemistry) edited by E.Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.

The polypeptides of the invention can also be prepared in solid phaseaccording to the methods described by Atherton and Shepard in their bookentitled “Solid phase peptide synthesis” (IRL Press, Oxford, 1989).

The polypeptides according to this invention can also be prepared bymeans of recombinant DNA techniques as described by Maniatis et al.,(Molecular Cloning: A Laboratory Manual, New York, Cold Spring HarborLaboratory, 1982).

According to another embodiment, the invention relates to a polynucleicacid comprising a sequence of at least 10 contiguous nucleotidesselected from:

(a) the polynucleic acid sequences which code for any of thepolypeptides described above, or

(b) the polynucleic acid sequences which are degenerate as a result ofthe genetic code to the polynucleic acid sequences as defined in (a),and which still encode a polypeptide as described above, or

(c) the polynucleic acid sequences which hybridize to any of thepolynucleic acids as defined in (a) or (b).

According to yet another embodiment, the present invention relates to apolynucleic acid sequence, in an isolated form, comprising a contiguoussequence of at least 10 nucleotides, more particularly 11, 12, 13, 14,15, 20 or more contiguous nucleotides selected from any of thepolynucleic acid sequences as described here above.

The term “polynucleic acid” refers to a single stranded or doublestranded nucleic acid sequence which may contain from 10 nucleotides tothe total number of nucleotides of the polynucleotide sequence (such asfor instance 20, 30, 40, 50, 60, 70, 80 or more nucleotides). Apolynucleic acid which is smaller than about 100 nucleotides in lengthis often also referred to as an oligonucleotide. A polynucleic acid mayconsist of deoxyribonucleotides or ribonucleotides, nucleotide analoguesor modified nucleotides, or may have been adapted for therapeuticpurposes. A polynucleic acid may also comprise a double stranded cDNAclone which can be used for cloning purposes, or for in vivo therapy, orprophylaxis.

The expression “hybridizes to” refers to preferably stringenthybridization conditions, allowing hybridisation between sequencesshowing at least 70%, 80%, 90%, 95% or more homology with each other.

The expression “in isolated form” refers to the fact that saidpolynucleic acid is preferably 90%, more preferably 95%, most preferably98% pure as measured by its weight versus the weight of possiblecontaminants.

The Brucella polynucleic acids according to this embodiment of thepresent invention are preferably more than 55% homologous, morepreferably more than 65%, and most preferably more than 75% homologous(e.g. more than 85%, more than 90%, more than 95% homologous) to thenucleic acid sequence shown in SEQ ID NO 1.

The terms “homologous” and “homology” are used in the current inventionas synonyms for “identical” and “identity”; this means that nucleic acidsequences which are e.g. said to be 55% homologous, show 55% identicalbasepairs in the same position upon alignent of the sequences.

According to a subsequent embodiment, the invention relates to apolynucleic acid as described above comprising a sequence of at least 10contiguous nucleotides selected from:

(a) the polynucleic acid sequence as shown in SEQ ID NO 1, or,

(b) the polynucleic acid sequences which are degenerate as a result ofthe genetic code to the polynucleic acid sequences as shown in SEQ ID NO1, and which still encode a Brucella abortus 17-kDa antigen as shown inSEQ ID NO 2,

(c) the polynucleic acid sequences which hybridize to the polynucleicacid sequences as defined in (a) or (b).

According to a preferred embodiment, the present invention relates toany nucleotide sequence which, upon expression, gives rise to apolypeptide sequence as represented in SEQ ID NO 2, or part thereof.

Another embodiment of the invention refers to a polynucleic acid asdescribed above being comprised in a cDNA or a genomic clone, with saidclone being obtainable by a process comprising essentially the followingsteps:

(a) preparing a Brucella species genomic library and/or expressionlibrary, by digestion of chromosomal DNA of said Brucella species andligation in an appropiate cloning and/or expression vector, and,

(b) immunoscreening said Brucella species expression library withBrucella field infected individuals sera, or with antibodies raisedagainst any of the polypeptides of the invention described above, and/or

(c) screening said Brucella species genomic library with a probehybridizing specifically with a polynucleic acid of the invention asdescribed above, and

(d) characterizing the positive clones obtained in the screening stepsof (b) and/or (c) by means of sequence analysis, after the insert of theobtained clones has been isolated and possibly amplified by means ofPCR, and

(e) possibly repeating step (c) and (d) using the obtained amplifiedmaterial as a probe to isolate the complete gene, and characterizing theobtained clone by means of sequence analysis.

The term “clone” refers to a population of cells or organisms formed byrepeated asexual division from a common cell or organism. To “clone agene” means to produce many copies of a gene by repeated cycles ofreplication.

The term “genomic library” refers to a collection of clones, each clonecontaining a different insert of (c)DNA in a cloning vector, said insertcorresponding to a different part of the genome of the respectiveorganism. A “(genomic) expression library” enables the expression of the(c)DNA inserts into polypeptide fragments.

Another embodiment of the invention provides for an oligonucleotideprobe comprising part of a polynucleic acid sequence as described above,with said probe being able to act as a specific hybridization probe fordetecting the presence of a Brucella polynucleic acid encoding any ofthe polypeptides as described above, or part thereof.

Preferably said oligonucleotide probe comprises at least 10 contiguousnucleotides, more preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 50 contiguous nucleotides selected from any of theabove-mentioned polynucleic acid sequences.

The term “probe” refers to single stranded sequence-specificoligonucleotides which have a sequence which is sufficientlycomplementary to hybridize to the target sequence to be detected.

Probes according to this aspect of the present invention may be chosenaccording to any of the techniques known in the art. Probes may beprovided hybridizing to Brucella species which cause brucellosis indifferent kinds of animals and in humans (B. abortus, B. melitensis, B.ovis, B. suis . . . ). More particularly, probes specificallyhybridizing to certain species of Brucella may be provided. Underappropriate hybridization conditions, such probes may allow todistinguish different Brucella species present in a sample to beanalyzed.

According to the hybridization solution (SSC, SSPE, etc.), these probesshould be stringently hybridized at their appropriate temperature inorder to attain sufficient specificity. However, by slightly modifyingthe DNA probes, either by adding or deleting one or a few nucleotides attheir extremities (either 3′ or 5′), or substituting some non-essentialnucleotides (i.e. nucleotides not essential to discriminate betweentypes) by others (including modified nucleotides or inosine) theseprobes or variants thereof can be caused to hybridize specifically atthe same hybridization conditions (i.e. the same temperature and thesame hybridization solution). Also changing the amount (concentration)of probe used may be beneficial to obtain more specific hybridizationresults. It should be noted in this context, that probes of the samelength, regardless of their GC content, will hybridize specifically atapproximately the same temperature in TMACl solutions (Jacobs et al.,1988).

The latter implies that variant probes contemplated within this aspectof the present invention can be defined as probes hybridizing with thesame specificity as the probe they are derived from, possibly underdifferent, but stringent, hybridization and wash conditions (differentsolutions, different concentrations of buffer, different concentrationsof probe, different temperatures).

The oligonucleotides used as primers or probes may also contain orconsist of nucleotide analoges such as phosphorothioates (Matsukura etal., 1987), alkylphosphorothiates (Miller et al., 1979) or peptidenucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or maycontain intercalating agents (Asseline et al., 1984).

As most other variations or modifications introduced into the originalDNA sequences of the invention, these variations will necessitateadaptions with respect to the conditions under which the oligonucleotideshould be used to obtain the required specificity and sensitivity.However, the eventual results of hybridization will be essentially thesame as those obtained with the unmodified oligonucleotides.

The introduction of these modifications may be advantageous in order topositively influence characteristics such as hybridization kinetics,reversibility of the hybrid-formation, biological stability of theoligonucleotide molecules. etc.

The term “complement” refers to a nucleotide sequence which is exactlycomplementary to an indicated sequence and which is able to hybridize tothe indicated sequences. It should be clear that all polynucleic acidsof the invention, although only represented by one strand, alsoencompass the other complementary strand. This implies that all probesand primers specified may also be used in their complementary form, beit under different hybridization or amplification conditions.

An oligonucleotide primer comprising part of a polynucleic acid sequenceas described above, with said primer being able to initiate specificamplification of a Brucella polynucleic acid encoding any of thepolypeptides of the invention as described above, or part thereof.

Preferably said oligonucleotide primer contains at least 10, morepreferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides of any of thepolynucleic acid sequences as described above.

The term “primer” refers to a single stranded DNA oligonucleotidesequence capable of acting as a point of initiation for synthesis of anextension product which is complementary to the nucleic acid strand tobe copied. The length and the sequence of the primer must be such thatthey allow to prime the synthesis of the extension products. Preferablythe primer is about 5-50 nucleotides long, more preferably 10-30nucleotides long. Specific length and sequence will depend on thecomplexity of the required DNA or RNA targets, as well as on theconditions of primer use such as temperature and ionic strength.

The fact that amplification primers do not have to match exactly withthe corresponding template sequence to warrant proper amplification isamply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al.,1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-basedamplification (NASBA; Guatelli et al., 1990; Compton, 1991),transcription-based amplification system (TAS; Kwoh et al., 1989),strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992)or amplification by means of Qβ replicase (Lizardi et al., 1988; Lomeliet al., 1989) of any other suitable method to amplify nucleic acidmolecules using primer extension. During amplification, the amplifiedproducts can be conveniently labelled either using labelled primers orby incorporating labelled nucleotides. Labels may be isotopic (³²P, ³⁵S,etc.) or non-isotopic (biotin, digoxigenin, etc.). The amplificationreaction is repeated between 20 and 80 times, advantageously between 30and 50 times.

According to a further embodiment, the invention relates to arecombinant vector particularly for cloning and/or expression of any ofthe polynucleic acids of the invention as described above, with saidrecombinant vector comprising a vector sequence and at least part of aany of the polynucleic acid sequence as described above, and wherein, incase of a expression vector, the coding sequence of said polynucleicacid sequence is operably linked to a control sequence comprised in thevector sequence and capable of providing for the expression of thecoding sequence by the specific host.

The expression “operably linked” refers to a juxtaposition wherein thecomponents are configured so as to perform their usual function. Thus,control sequences operably linked to a coding sequence are capable ofeffecting the expression of the coding gene.

The term “control sequences” refers to those sequences which control thetranscription and/or translation of the coding sequences; these mayinclude but are not limited to the promoter sequences, transcriptionaland translational initiation (ribosome binding sites) and terminationsequences. In addition, control sequences refer to sequences whichcontrol the processing of the polypeptide encoded within the codingsequence; these may include, but are not limited to sequences controlingsecretion, protease cleavage, and glycosylation of the polypeptide.

The term “recombinant vector” may include a plasmid, a phage, a cosmidor a virus. A variety of vectors may be used to obtain recombinantexpression of antigenic proteins. Bacteria are most often transformed byplasmids or bacteriophages. Lower eukaryotes such as yeasts aretypically transformed with plasmids, or are transformed with arecombinant virus. The vectors may replicate within the hostindependently, or may integrate into the host cell genome. Highereukaryotes may be transformed with vectors, or may be infected with arecombinant virus, for example a recombinant vaccinia virus.

According to an alternative embodiment, the current invention alsoprovides for a recombinant vector particularly for cloning and/orexpression of heterologous sequences, with said recombinant vectorcomprising a vector sequence and the control elements, or parts thereof,comprised in the polynucleic acids of the invention, and wherein, incase of an expression vector, said control elements are operably linkedto the coding sequence of the heterologous gene to be expressed.

The term “heterologous sequence” as used in the current inventionsignifies any sequences different from the 17 kDa Brucella antigensequences. Said heterologous sequences may also be called “foreign”sequences.

The control sequences comprised in the polynucleic acids of theinvention are located in the region upstream of the sequences coding forthe polypeptides of the invention.

The invention further relates to a host cell transformed by any of therecombinant vectors described above, with said host cell beingpreferably a prokaryotic organism, and more preferably E. coli, aSalmonella species or a lactic acid bacterium.

Host cells suitable for the expression of the polynucleic acids of theinvention may also include lower eukaryotic cells (like yeasts) orhigher eukaryotic cells.

Another embodiment of the invention provides for a recombinantpolypeptide encoded by at least part of any of the polynucleic acids ofthe invention described above, and being expressed in a transformedcellular host as described here above.

Said recombinant polypeptide is also called an “expression product”.

The invention further relates to a recombinant polypeptide as describedabove, with said recombinant polypeptide consisting of a heterologoussequence, provided by the vector, fused in frame to the amino acidsequence of any of the polypeptides of the invention described above orpart thereof.

Said recombinant polypeptide fused to a heterologous sequence is alsocalled a “fusion protein”. The heterologous sequence may bring about anydesired side effect to the resulting fusion protein, e.g. it mayoptimize the expression, the purification, the immobilization on asurface etc.

The invention also relates to a method for production of a recombinantpolypeptide as described above, comprising:

transformation of an appropiate cellular host with a recombinantexpression vector as described above, wherein any of the polynucleicacids of the invention, or part thereof, has been inserted under thecontrol of the appropiate regulatory elements,

culturing said transformed cellular host under conditions enablingexpression of said insert, and

harvesting and purifying said polypeptide.

In order to carry out the expression of the polypeptides of theinvention in bacteria, like E. coli, the above-mentioned steps can befollowed according to principles known in the art, as examplified in theExamples section.

The techniques for carrying out the expression of recombinantpolypeptides in any of the other hosts as specified above, are also wellknown in the art of recombinant expression technology.

The invention further relates to an antibody recognizing specificallyany of the polypeptides of the invention as described above, with saidantibody being possibly a polyclonal antibody, and preferably amonoclonal antibody.

Preferably, said monoclonal antibody of the invention is different fromA66/05H01/E09 (Cloeckaert et al., 1991).

A further embodiment of the present invention relates to an antibody,more particularly a monoclonal antibody, characterized in that it isspecifically raised against an antigenic determinant of an isolated17-kDa Brucella polypeptide.

According to an alternative embodiment, the present invention alsorelates to an antigen-binding fragment of the antibody, said fragmentbeing of the F(ab′)₂, Fab or single chain Fv type, or any type ofrecombinant antibody derived from said specific antibodies or monoclonalantibodies.

The terms “antigenic determinant” or “epitope” refer to that portion ofa molecule that is specifically bound by an antibody combining site.Antigenic determinants may be determined by any of the techniques knownin the art or may be predicted by a variety of computer predictionmodels known in the art.

The expression “antibody recognizing specifically” means that bindingbetween the antigen as a ligand and a molecule containing an antibodycombining site, such as a Fab portion of a whole antibody, is specific,signifying that no cross-reaction occurs.

The expression “antibody specifically raised against a compound” meansthat the sole immunogen used to produce said antibody was said compound.

Antibodies according to a preferred embodiment of the invention includespecific polyclonal antisera raised against the Brucella polypeptides ofthe invention, and having no cross-reactivity to others proteins, ormonoclonal antibodies raised against the Brucella polypeptides of theinvention.

The possible crosseactivity of the polyclonal antisera may be eliminatedby preabsorption of the polyclonal antiserum against the cross-reactingantigenic determinants.

The monoclonal antibodies of the invention can be produced by anyhybridoma liable to be formed according to classical methods fromsplenic cells of an animal, particularly of a mouse or rat, immunizedagainst the adhesive polypeptides according to the invention, definedabove on the one hand, and of cells of a myeloma cell line on the otherhand, and to be selected by the ability of the hybridoma to produce themonoclonal antibodies recognizing the polypeptides which have beeninitially used for the immunization of the animals.

The monoclonal antibodies according to this preferred embodiment of theinvention may be humanized versions of the mouse monoclonal antibodiesmade by means of recombinant DNA technology, departing from the mouseand/or human genomic DNA sequences coding for H and L chains or fromcDNA clones coding for H and L chains.

Also fragments derived from these monoclonal antibodies such as Fab,F(ab)′₂ and ssFv (“single chain variable fragment”), providing they haveretained the original binding properties, form part of the presentinvention. Such fragments are commonly generated by, for instance,enzymatic digestion of the antibodies with papain, pepsin, or otherproteases. It is well known to the person skilled in the art thatmonoclonal antibodies, or fragments thereof, can be modified for varioususes.

The antibodies involved in the invention can be labelled by anappropriate label of the enzymatic, fluorescent, or radioactive type.

The invention also relates to the use of the proteins of the invention,muteins thereof, or fragments thereof, for the selection of recombinantantibodies by the process of repertoire cloning (Perrson et al., 1991).

According to a preferred embodiment of the present invention, anantibody, or an antigen-binding fragment F(ab′)₂, Fab, single chain Fvand all types of recombinant antibodies, as defined above are furthercharacterized in that they can inhibit the infection of Brucella strainsto the specific cell type which they infect in vivo.

According to another embodiment, the present invention relates to amonoclonal antibody as defined above, obtainable by a process comprisingat least the following steps:

fusing the splenocytes from mice infected with Brucella species togetherwith myeloma cells, and

selecting the anti-Brucella hybridomas by means of ELISA and subsequentlimiting dilution,

selecting the hybridomas producing a monoclonal antibody, specificallydirected against any of the 17-kDa Brucella polypeptides by means ofELISA, and,

recovering the monoclonal antibodies from ascites fluid or from aculture of the selected hybridomas.

The present invention also relates to a hybridoma producing any of themonoclonal antibodies as defined above.

The present invention further relates to an anti-idiotype antibodyraised against any of the antibodies as defined above.

The term “anti-idiotype antibodies” refers to monoclonal antibodiesraised against the antigenic determinants of the variable region ofmonoclonal antibodies themselves raised against the Brucella 17 kDapolypeptides. These antigenic determinants of immunoglobulins are knownas idiotypes (sets of idiotopes) and can therefore be considered to bethe “fingerprint” of an antibody (for review see de Preval, 1978;Fleishmann and Davie. 1984). The methods for production of monoclonalanti-idiotypic antibodies have been described by Gheuens and McFarlin(1982). Monoclonal anti-idiotypic antibodies have the property offorming an immunological complex with the idiotype of the monoclonalantibody against which they were raised. In this respect the monoclonalantibody is often referred to as Ab1, and the anti-idiotypic antibody isreferred to as Ab2. These anti-idiotype Ab2s may be used as substitutesfor the polypeptides of the invention or as competitors for binding ofthe polypeptides of the invention to their target.

The present invention further relates to antisense peptides derived fromthe Brucella polypeptides as defined above.

More particularly, the term “antisense peptide” is reviewed by Blalock(1990) and by Roubos (1990). In this respect, the molecular recognitiontheory (Blalock, 1990) states that not only the complementary nucleicacid sequences interact but that, in addition, interacting sites inproteins are composed of complementary amino acid sequences (senseligand with receptor or sense ligand with antisense peptides). Thus, twopeptides derived from complementary nucleic acid sequences in the samereading frame will show a total interchange of their hydrophobic andhydrophilic amino acids when the amino terminus of one is aligned withthe carboxy terminus of the other. This inverted hydropathic patternmight allow two such peptides to assume complementary conformationsresponsible for specific interaction.

The antisense peptides can be prepared as described in Ghiso et al.(1990). By means of this technology it is possible to logicallyconstruct a peptide having a physiologically relevant interaction with aknown peptide by simple nucleotide sequence analysis forcomplementarity, and synthesize the peptide complementary to the bindingsite.

The present invention still further relates to a method for in vitrodiagnosis of Brucella (species) infection comprising at least the stepof contacting a sample possibly containing anti-Brucella (species)17-kDa polypeptide antibodies, Brucella (species) 17-kDa antigens and/orBrucella (species) 17 kDa protein encoding nucleic acids, with:

a 17-kDa Brucella polypeptide or peptide as defined above, underconditions allowing the formation of an immunological complex, or,

a Brucella 17-kDa polynucleic acid sequence as defined above, underconditions allowing the formation of a hybridization complex, with saidnucleic acids of said sample being possibly amplified prior tohybridization, or,

an antibody specifically directed against a 17-kDa antigen as definedabove, under conditions allowing the formation of an immunologicalcomplex, or,

an anti-idiotype antibody as defined above, under conditions allowingthe formation of an antibody-anti-idiotypic complex, or,

an antisense peptide as defined above, under conditions allowing theformation of an antigen-antisense peptide complex,

and subsequently detecting the complexes formed.

The Brucella species which may be diagnosed by the above method includeB. abortus, B. melitensis, B. ovis, B. suis.

The term “sample” may refer to any biological sample (tissue or fluid)containing Brucella nucleic acid sequences, antibodies or polypeptides.

A preferable detection method is the detection of antibodies againstBrucella (species), and the preferred sample in that case is serum orplasma.

In a more specific embodiment, the invention relates to a method fordetecting antibodies to Brucella (species) present in a biologicalsample, comprising:

contacting the biological sample to be analysed with any of thepolypeptide as described above, under conditions allowing the formationof an immunological complex, and

detecting the immunological complex formed between said antibodies andsaid polypeptide.

Conditions allowing the formation of an immunological complex are knownto the person skilled in the art.

In a special embodiment, the polypeptides being used in theabove-described method for detection of anti-Brucella speciesantibodies, can be replaced by anti-idiotype antibodies as describedabove, acting as their equivalents.

Conditions allowing the formation of an antibody-anti-idiotypic complexare known in the art.

The invention further relates to a method for detecting the presence ofBrucella (species) antigens in a biological sample, comprising:

contacting the biological sample to be analysed with an antibodyaccording to the invention, under conditions allowing the formation ofan immunological complex, and

detecting the immunological complex formed between said antigens andsaid antibody.

In a special embodiment, the antibodies being used in theabove-described method for detection of Brucella antigens, may bereplaced by anti-sense peptides as described above, acting as theirequivalents.

Conditions allowing the formation of an antigen-antisense peptidecomplex are known in the art.

Design of immunoassays is subject to a great deal of variation, and manyformats are known in the art. Protocols may, for example, use solidsupports, or immunoprecipitation. Most assays involve the use of labeledantibody or polypeptide; the labels may be, for example. enzymatic,fluorescent, chemiluminescent, radioactive, or dye molecules. Assayswhich amplify the signals from the immune complex are also known;examples of which are assays which utilize biotin and avidin orstreptavidin, and enzyme-labeled and mediated immunoassays, such asELISA assays.

An advantageous embodiment provides for a method for detection ofanti-Brucella (species) antibodies in a sample, whereby the polypeptidesof the invention are immobilized on a solid support, eventually on amembrane strip. Different polypeptides of the invention may beimmobilized together or next to each other (e.g. in the form of parallellines). The polypeptides of the invention may also be combined withother antigens from other organisms, belonging to the genus Brucella orto other genera.

The combination of different antigens in one single detection method asdescribed may have certain advantages, such as:

achieving a higher test sensitivity: e.g. by combining several antigenicdeterminants from Brucella species, the number of positively reactingsera from field infected individuals may be greater, and/or

enabling differentiation between individuals infected by differentpathogens, belonging to the genus Brucella or to other genera,

enabling differentiation between Brucella (species) field-infected andvaccinated individuals.

The invention thus also relates to a solid support onto which thepolypeptides of the invention, possibly in combination with otherpolypeptides, have been immobilized.

Another embodiment of the invention provides for a method for detectingthe presence of Brucella (species) polynucleic acids present in abiological sample, comprising:

possibly extracting the polynucleic acids contained in the sample,

amplifying the Brucella (species) polynucleic acids with at least oneprimer as described above,

detecting the amplified nucleic acids, possibly after hybridization witha probe as described above.

Conditions allowing hybridization are known in the art and e.g.exemplified in Maniatis et al. (1982). However, according to thehybridization solution (SSC, SSPE, etc.), the probes used should behybridized at their appropriate temperature in order to attainsufficient specificity (in some cases differences at the level of onenucleotide mutation are to be discriminated).

Amplification of nucleic acids present in a sample prior to detection invitro may be accomplished by first extracting the nucleic acids presentin the sample according to any of the techniques known in the art. Incase of extraction of RNA, generation of cDNA is necessary; otherwisecDNA or genomic DNA is extracted.

The amplification methods are detailed above.

Suitable assay methods for purposes of the present invention to detecthybrids formed between oligonucleotide probes according to the inventionand the nucleic acid sequences in a sample may comprise any of the assayformats kown in the art. For example, the detection can be accomplishedusing a dot blot format, the unlabelled amplified sample being bound toa membrane, the membrane being incubated with at least one labelledprobe under suitable hybridization and wash conditions, and the presenceof bound probe being monitored. Probes can be labelled withradioisotopes or with labels allowing chromogenic or chemilumeniscentdetection such as horse-radish peroxidase coupled probes.

An alternative is a “reverse” dot-blot format, in which the amplifiedsequence contains a label. In this format, the unlabelledoligonucleotide probes are bound to a solid support and exposed to thelabelled sample under appropriate stringent hybridization and subsequentwashing conditions. It is to be understood that also any other assaymethod which relies on the formation of a hybrid between the nucleicacids of the sample and the oligonucleotide probes according to thepresent invention may be used.

According to an advantageous embodiment, the process of detectingBrucella polynucleic acid sequences contained in a biological samplecomprises the steps of contacting amplified copies derived from thegenetic material, with a solid support on which probes as defined above,have been previously immobilized.

In a very specific embodiment, the probes have been immobilized on amembrane strip in the form of parallel lines. This type of reversehybridization method is specified further as a Line Probe Assay (LiPA).

The invention thus also relates to a solid support onto which thepolynucleotides of the invention have been immobilized.

The invention further relates to a method for detecting individualshaving been in contact with Brucella (species), comprising:

contacting a polypeptide according to the invention with the cellularimmune system of the individual, either in vitro or in vivo, and

detecting and quantifying the cellular immune response raised againstsaid polypeptides.

The above-said cellular immune response can be measured either in vivo,such as a delayed type hypersensitivity reaction upon subcutaneousinjection of the polypeptides of the invention, or in vitro, such asstimulation of periferal blood lymphocytes or secretion ofinterferon-gamma, upon addition of the polypeptides of the invention toa sample of periferal blood lymphocytes under conditions allowingrecognition of the polypeptides by the cells responsive for the immuneresponse, conditions which are known to the person skilled in the art.

The invention further relates to a diagnostic kit for the detection ofantibodies to Brucella (species) present in a biological sample, saidkit comprising any of the polypeptides according to the invention, withsaid polypeptides being preferably bound to a solid support.

The present invention relates more particularly to a kit for determiningthe presence of anti-Brucella (species) antibodies as defined abovepresent in a biological sample liable to contain them, comprising:

at least one polypeptide or peptide as defined above, preferentially incombination with other polypeptides or peptides from Brucella, with saidpolypeptides being preferentially immobilized on a solid substrate,

a buffer or components necessary for producing the buffer enablingbinding reaction between these polypeptides and the antibodies againstthe Brucella 17 kDa protein present in the biological sample,

means for detecting the immune complexes formed in the preceding bindingreaction,

possibly also including an automated scanning and interpretation devicefor inferring the presence of Brucella antibodies in the sample from theobserved binding pattern.

The kit according to this aspect of the present invention may comprisein addition to peptide or polypeptide antigens according to theinvention, also other Brucella antigenic proteins or peptides known inthe art (such as outer membrane protein (OMP) proteins or peptides), orother bacterial antigenic proteins or peptides in general.

In a very specific embodiment the invention relates to a kit for thedetection of anti-Brucella species antibodies in a biological sample asdescribed above, whereby the polypeptides of the invention are replacedby the anti-idiotype antibodies as described above.

The invention further relates to a diagnostic kit for the detection ofantigens of Brucella (species) present in a biological sample, said kitcomprising an antibody as described above, with said antibody beingpreferably bound to a solid support.

In a very specific embodiment, the invention relates to a diagnostic kitfor the detection of antigens of Brucella species present in abiological sample, whereby the antibody as described above is replacedby an antisense peptide.

The invention further also relates to a diagnostic kit for the detectionof Brucella (species) polynucleic acids present in a sample, said kitcomprising a probe as described above and/or a primer as describedabove.

According to a preferred embodiment, the present invention relates to akit for determining the presence of Brucella polynucleic acids asdefined above present in a biological sample liable to contain them,comprising:

possibly at least one primer or a set of primers as defined above,

at least one oligonucleotide probe as defined above, with said probesbeing preferentially immobilized on a solid substrate, and morepreferentially on one and the same membrane strip,

a buffer or components necessary for producing the buffer enabling ahybridization reaction between these probes and the possibly amplifiedproducts to be carried out,

a solution or components necessary for producing the solution, enablingwashing of the hybrids formed under the appropiate wash conditions,

means for detecting the hybrids resulting from the precedinghybridization,

possibly also including an automated scanning and interpretation devicefor inferring the Brucella (strain) polynucleic acids present in thesample from the observed hybridization pattern.

According to this advantageous method, the probes are immobilized in aLine Probe Assay (LiPA) format. This is a reverse hybridization format(Saiki et al., 1989) using membrane strips onto which severaloligonucleotide probes (including negative or positive controloligonucleotides) can be conveniently applied as parallel lines.

The invention thus also relates to a solid support, preferably amembrane strip, carrying on its surface one or more probes as definedabove, coupled to the support in the form of parallel lines.

A LiPA support may contain on its surface different oligonucleotideprobes derived from polynucleic acid sequences according to theinvention which hybridize specifically with certain strains of Brucella(such as B. abortus, B. melitensis, B. ovis, B. suis) or may contain atleast one Brucella oligonucleotide probe derived from a polynucleic acidsequence according to the present invention in addition to otherBrucella probes or probes derived from other bacterial and/or viralorganisms.

The LiPA is a very rapid and user-friendly hybridization test. Resultscan be read 4 h after the start of the amplification. Afteramplification during which usually a non-isotopic label is incorporatedin the amplified product, and alkaline denaturation, the amplifiedproduct is contacted with the probes on the membrane and thehybridization is carried out for about 1 to 1,5 h hybridized polynucleicacid is detected. From the hybridization pattern generated, the Brucellastrain can be deduced either visually, but preferably using dedicatedsoftware. The LiPA format is completely compatible with commerciallyavailable scanning devices, thus rendering automatic interpretation ofthe results very reliable. All those advantages make the LiPA formatliable for the use of Brucella detection in a routine setting. The LiPAformat should be particularly advantageous for detecting the presence ofdifferent Brucella strains.

The invention further relates to a kit for the detection andquantification of the cellular immune response against Brucella(species) in an individual, said kit comprising any of the polypeptidesaccording to the invention as described above.

A method and kit for diagnosis, based on the quantification of thecellular immune response, as specified above, enables the identificationof individuals (humans or ruminants) which have been in contact with theBrucella pathogen. Said contact may subsequently lead to a disease state(=field infected individuals) or to a protected state of the individual(=vaccinated individuals).

According to a preferred embodiment, the present invention relates to amethod or a kit for diagnosis of Brucella infection as defined above,further characterized in that said polypeptides, peptides, polynucleicacids, antibodies, anti-idiotypic antibodies or anti-sense peptides areparticularly useful for differentiating Brucella (species) fieldinfected individuals from Brucella vaccinated individuals.

The invention further relates to a vaccine composition which providesprotective immunity against Brucella (species) infection in a mammal(human, ruminants) comprising as an active principle at least one of thepolypeptides according to the invention, or at least one of thepolynucleic acid sequences or recombinant vectors according to theinvention, said active principle being combined with a pharmaceuticallyacceptable carrier.

According to a special embodiment, the vaccine composition as describedabove may comprise as an active principle one of the anti-idiotypeantibodies as described above.

Besides the Brucella 17 kDa proteins according to the invention, saidvaccine composition may comprise also any other Brucella immunogeniccomponent (such as outer membrane proteins (OMP) Cloeckaert et al. 1991)or any other bacterial or other immunogenic component in general.

In a specific embodiment, polynucleic acid sequences coding for any ofthe polypeptides as defined above, are used as a vaccine, either asnaked DNA or as part of recombinant vectors. In this case, it is the aimthat said nucleic acids are expressed into immunogenic protein/peptideand thus confer in vivo protection to the vaccinated host (e.g. Ulmer etal., 1993).

The active ingredients of such a vaccine composition may be administeredorally, subcutaneously, conjunctivally, intramuscularly, intranasally,or via any other route known in the art including for instance via thebinding to carriers, via incorporation into liposomes, by addingadjuvants known in the art, etc.

According to another embodiment, the current invention also provides fora recombinant Brucella (species) strain in which the gene encoding aBrucella 17-kDa antigen as described above has been deleted orinactivated.

The invention also relates to a vaccine composition which providesprotective immunity against Brucella (species) infection in a mammal(human, ruminants) and which enables the differentiation between fieldinfected and vaccinated individuals, said vaccine composition comprisingas an active principle a recombinant Brucella (species) strain in whichthe gene encoding a Brucella 17 kDa antigen has been deleted orinactivated. The present embodiment is illustrated further in Example 7.

The above-described recombinant Brucella strain may be an interestingcomponent of a vaccine composition, providing protective immunityagainst brucellosis, yet permitting differentiation between Brucellafield infected and vaccinated individuals, because of its distinctiveimmunological signature.

The invention also relates to any of the above-mentioned substances(polypeptides, antibodies, polynucleic acids, anti-idiotype antibodies,antisense peptides) for use as a medicament, more particularly for anyof the medical (diagnostic or prophylactic) applications as mentionedabove.

Furthermore, the invention relates to the use of any of theabove-mentioned substances (polypeptides, antibodies, polynucleic acids,anti-idiotype antibodies, antisense peptides) for the manufacture of amedicament, more particularly for the preparation of a vaccine or forthe preparation of a diagnostic composition.

FIGURE AND TABLE LEGENDS

Table 1: Amino acid substitutions which may form the basis of themuteins according to the present invention.

Table 2: Sheep sera and cow sera were tested by Western blot experimentson total B. melitensis and B. abortus protein extract, respectively. Thesheep naturally infected but negative in classical serology wereconsidered positive upon a positive DTH reaction. Ten cows werevaccinated and for 3 of them the vaccination was successful (P), for theother seven animals, protection was not obtained (NP).

Most of these sera were then retested on B. abortus total proteinextract in competitive ELISA with a monoclonal antibody specific for the17-kDa antigen. *: number of positive sera (a band at 17 kDa in Westernblots or at least 30% inhibition in ELISA).

Table 3: Summary of the seroreactivity of different species (goat,sheep, cattle) with the 17 kDa Brucella abortus protein. n=number ofanimals tested.

FIG. 1 Nucleotide sequence (SEQ ID NO 1) of the 811 bp fragment encodingthe 17-kDa Brucella abortus protein. The predicted amino acid sequence(SEQ ID NO 2) is shown below the nucleotide sequence. The putative Shineand Delgarno sequence and the stop codon are shown in bold.

FIG. 2 Western blot analysis of B. melitensis proteins. Blots wereprobed with a sheep serum (Lane 1) or with monoclonal antibodiesdirected against the OMP16.5 as described by Cloeckaert et al. (1991)(A68/04G01/C06, Lane 2) or directed against the 17 kDa protein of thisinvention (A66/05H01/E09, Lane 3). A: B. melitensis total proteinextract (rough strain H38, 40 μg/well); B: purified-recombinant OMP16.5(0.1 μg/well); C: extract from recombinant E. coli expressing the 17-kDaantigen (40 μg/well); D: Extract from E. coli control strain.

FIG. 3 Protein sequence of the 17-kDa fusion protein as produced in E.coli (SEQ ID NO 3). The Brucella sequence is shown in bold.

FIG. 4: Reactivity of a set of individual goat sera on microwells coatedwith the recombinant 17 kDa Brucella abortus antigen. The horizontalline indicates the cutoff value, calculated as the mean of the negativesera plus 3 SD. Negative goat sera were obtained from a region free ofbrucellosis.

FIG. 5: Reactivity of a set of human sera from Brucella infectedpatients on microwells coated with the recombinant 17 kDa Brucellaabortus antigen. The cutoff value was calculated as the mean of thenegative sera (blood donors) plus 3 SD.

EXAMPLES MATERIALS AND METHODS

Reagents. All reagents were of analytical grade and obtained from Merck(Darmstadt, Germany), Sigma (St. Louis, Mo.), or Bio-Rad Laboratories(Richmond, Calif.). Restriction enzymes and DNA modifying enzymes werepurchased from Boehringer Mannheim (Brussels, Belgium) and were usedaccording to the manufacturer's instructions. Protein concentrationswere determined by the bicinchoninic acid (Pierce, Rockford, Ill.).

Bacteria and vectors. E. coli Y1090, E. coli MC1061, E. coli DH5 alphaF′ competent cells and pBluescript SK+ vector were from Stratagene (LaJolla, Calif.).

Monoclonal antibodies. The monoclonal antibodies (Mabs A66/05H01/E09 andA68/04G01/C06) were prepared as described in Cloeckaert et al., 1990 andCloeckaert et al., 1991. Ascitic fluids or hybridoma culturesupernatants were used.

Sera. Sheep sera were obtained from J. Blasco (Servicio de InvestigacionAgraria, Zaragoza, Spain) and cow sera were from the FacultéUniversitaire Notre-Dame de la Paix (FUNDP). One hundred sera fromnaturally B. melitensis-infected sheep and 36 cow sera from naturally B.abortus-infected animals with a positive classical serology for Brucellainfection (Alton et al., 1988) were used. Twenty sheep sera frominfected animals which had a negative serology but a positive DTHreaction were also included. Sera from experimentally vaccinated andinfected cows were obtained as follows. Pregnant heifers (10 animals)were vaccinated subcutaneously with B. abortus strain B19 (150×10⁶ CFU).At 88 days post-vaccination, the heifers were conjunctively infectedwith 16.6×10⁶ B. abortus strain 544. Animals were bled 135 dayspost-infection. Fifteen sheep sera and 14 cow sera from healthy animalswere used as controls. Successful vaccination was proven by the absenceof infection of the heifers and of the new born calves.

Preparation of bacterial protein extracts. Total protein extract from B.melitensis strain H38R (depleted in high molecular weightlipopolysaccharide) was kindly provided by G. Dubray (INRA, Nouzilly,France). Briefly, the bacteria were grown on solid agar, collected bywashing and heated at 95° C. for 1 h in Laemmli buffer (Laemmli, 1970)containing 2% SDS. Total protein extract from B. abortus 45/20 roughstrain were grown in liquid medium, centrifuged and treated as above.Total protein extract from recombinant E. coli was obtained from 5 mlovernight culture (O.D. at 600 nm=1) in Luria broth medium (L. B.,Maniatis et al., 1982) supplemented with 0.1 g/l ampicillin and 1%glucose. Bacteria were centrifuged for 10 min at 5,000×g. The pellet wasresuspended in 1 ml of electrophoresis sample buffer (Laemmli, 1970),heated at 100° C. for 5 min. and centrifuged.

Recombinant B. abortus minor outer membrane protein (OMP). The OMP16.5gene (Tibor et al. , 1994) was expressed in E. coli as a fusion proteinwith a mTNF leader peptide (25 amino acids, Van Gelder et al., 1993) andbetween the mTNF and the OMP, a cluster of 6 histidine residues wasinserted to allow purification of the fusion protein by immobilizedmetal-ion affinity chromatography (IMAC, Hochuli et al., 1988). Thefusion protein was purified to at least 99% homogeneity, as determinedby gel electrophoresis followed by silver staining (unpublishedresults). Due to this fusion, this recombinant protein migrated at anapparent molecular weight of 20 kDa in SDS-PAGE.

Construction of a B. abortus genomic library. The lambda gtl 1 B.abortus genomic library was generously provided by Dr. P. de Wergifosse(Université catholique de Louvain, Belgium, de Wergifosse, 1992). It wasprepared by Sau3A digestion of B. abortus chromosomal DNA. The stickyends were filled-in, and EcoR1 linkers were added to insert the DNAfragments into the lambda gt11 EcoR1 cloning site. General molecularbiological techniques were performed as described by Maniatis et al.(1982).

Library plating and immunoscreening. A bacteriophage suspension (10 μl)in phage dilution buffer (20 mM Tris, 100 mM NaCl, 10 mM magnesiumsulfate, pH 7.4) was incubated (20 min at 37° C.) with an overnightculture (0.6 ml) of E. coli Y1090, grown at 37° C. in LB andsupplemented with 0.1 g/l ampicillin, 10 mM magnesium sulfate and 2%maltose. Thirty ml of top agar (LB+0.7% agar+0.1 g/l ampicillin+10 mMmagnesium sulfate) prewarmed at 48° C., was then added to the mixtureand immediately plated onto a 220×220 mm Petri dish containing 150 ml ofsolid medium (LB containing 1.2% agar). After plating, the dishes wereleft for 5 h at 37° C. and subsequently covered with a nitrocellulosemembrane (Hybond-C; Amersham, Brussels, Belgium), wetted in a 10 mMisopropyl β-D-thiogalactopyranoside (IPTG) solution, and blotted drybetween two sheets of 3 MM Whatman paper. After overnight incubation(37° C.), the membrane was peeled off and saturated for 1 h at roomtemperature with 5% fat-free milk in a Tris salt buffer (TBS) pH 7.4containing 0.05% NP40 (TBS is 10 mM Tris and 150 mM NaCl). Allsubsequent incubations were also performed at room temperature. Serafrom B. melitensis-infected sheep were diluted 1/40 in TBS-NP40 buffer,containing 5% fat-free milk and E. coli MC1061 lysate (diluted to 1mg/ml of protein content) and the mixture was incubated with themembrane for 90 min. After 3 washes in TBS-NP40, a rabbit anti-sheepalkaline phosphatase conjugate (Jackson, West Grove, Pa.) diluted 1/5000in TBS-NP40 was added to the membrane for a 1 h incubation. After 3 morewashes, bound antibody was revealed with NBT/BCIP (0.37 mM each) in aTris buffer pH 9.5 (0.1 M Tris, 0.1 M NaCl, 5 mM magnesium sulfate) for10 min. A positive plaque was removed from the top agar with a steriletip and suspended in 100 μl phage dilution buffer. The positivebacteriophage was replated several times until plaque lifts showed morethan 99% immunoreactive plaques.

Gel electrophoresis and Western blotting. The total bacterial proteinextracts (40 μg/well) were analyzed by SDS-PAGE (12.5%) in the presenceof β-mercapto ethanol as described by Laemmli (Laemmli, 1970). Proteinswere transferred by semi-dry Western blotting (Tsang et al., 1983)(transfer buffer: 25 mM Tris, 192 mM glycine, 20% methanol) tonitrocellulose for 40 min at 0.8 mA per cm². The membrane was saturatedwith 5% fat-free milk as describe above and incubated overnight withmonoclonals or sera. Ascites were diluted 1/1,000 in TBS-NP40 containing5% fat-free milk and sera were diluted 1/40 in TBS-NP40 containing 5%fat-free milk and E. coli lysate (final E. coli protein concentration: 1mg/ml). After 3 washes, bands were revealed with rabbit anti-mouse IgGconjugate (Prosan, Denmark), rabbit anti-sheep conjugate (as describedabove) or rabbit anti-cow conjugate (1/5000, Sigma, St Louis, Mo.).

Competitive ELISA. Sonicated cell extract of B. abortus (strain 45/20)was coated on microplates (69620, Nunc) at 20 μg/ml in 5 fold dilutedbuffer (GBS: 0.17 M NaCl, 0.1 M glycine and 6 mM NaN₃, pH 9.2). Aftersaturation with casein hydrolysate, twofold diluted sera and Mab ascite(1/10,000), in a GBS buffer supplemented with 50 mM EDTA and 0.1%Tween-20 (GBS-EDTA-Tw), were incubated for 1 h at room temperature inthe microwells. The binding of the Mab was revealed by 1 h incubation atroom temperature with a sheep anti-mouse peroxidase-conjugate (Amersham,Brussels, Belgium) diluted 1/1000 in GBS-EDTA-Tw containing 4% caseinhydrolysate. Reagents in excess were removed between each step by 6washings with a 0.15 M NaCl solution containing 0.01% Tween-20.O-Phenylenediamine (0.4% wt/vol) with 2 mM H₂O₂ in a citrate-phosphatebuffer (0.05 M Na₂HPO₄, 0.025 M citric acid, pH 5) was used to developthe assay. The signal was read at 490 nm and 630 nm and the differencewas recorded using a BIO kinetics Reader EL-340 (Bio-tek instruments,inc., Vermont, USA). A reduction in signal of more than 30% relative tothe serum free control, was considered as a positive competitionreaction.

Colony blotting. Recombinant E. coli colonies grown on solid medium weretransferred by colony blotting to a nitrocellulose disc prewetted in PBS(135 mM NaCl, 10 mM Na₂HPO₄, 1 mM KH₂PO₄, 5 mM KCl, pH 7.4). The filterwas gently shaken in a solution containing PBS with 0.5% Tween-20 for 30min at room temperature to lyse the bacteria. The membrane wassubsequently rinsed twice with TBS-NP40 and the immunoscreening wasperformed as described above.

Polymerase Chain Reaction (PCR; Mullis & Falnoona, 1987). The B. abortusDNA insert was recovered from the purified bacteriophage by PCR directlyon the isolated bacteriophage. With lambda gt11 primers (Stratagene, LaJolla, Calif.) the following conditions were used: The reaction mixture(50 μtl) contained 0.1 M PFU of recombinant phage, 100 pmol of eachprimer, 200 mM of each dNTP (Pharmacia, Uppsala Sweden), 1 unit of Taqpolymerase (Cetus, Emeryville, Calif.) with the appropriate buffer. Thereaction mixture was heated for 5 min at 95° C. and then 40 cycles (95°C., 55° C. and 72° C., 1 min each) were performed in a thermocycler(Perkin Elmer Cetus, Emeryville, Calif.). To terminate, a 10 minelongation step at 72° C. was added.

Cloning. DNA fragments, obtained by EcoR1 or HindIII digestion, werecloned in a pBluescript SK+ vector (SK+, Stratagene, La Jolla, Calif.)and plasmids were transformed and propagated in E. coli DH5 alpha F′.Plasmid purification was performed on a QIAGEN matrix (Diagen, Germany)as described by the manufacturer.

Nucleic acid sequencing. Sequence analysis of the DNA fragments clonedin SK+ plasmids was performed using the chain termination procedure(Sanger et al., 1977), adapted to allow analysis on an automated DNAsequencer (Applied Biosystems, Foster city, Calif.). Sequencingreactions were carried out using the dye-terminator technology, asdescribed by the manufacturer, using the universal or reverse M13primers. In order to obtain the complete sequence of the fragment, 2internal oligonucleotides were custom synthesized (Phamacia, Uppsala,Sweden) to enable internal priming. Sequence manipulations wereperformed using the Intelligenetics software package (California, USA).

Example 1 Cloning and Sequencing of the B. abortus Gene Encoding a17-kDa Antigen Identification of a New Gene

In a first screening, about 35,000 gt11 plaques of the lambda gt11 B.abortus genomic library (prepared as described in the Materials andMethods section) were probed with a pool of field sera from B.melitensis-infected sheep. One positive plaque was identified andpurified by repeated plating. The purified bacteriophage was againplated and the plaques were probed with different Mabs directed to B.abortus proteins by means of an immunoscreening as described in theMaterials and Methods section (Cloeckaert et al., 1990, 1991). One Mab(A66/05H01/E09) clearly yielded a strong signal with the selected phage.To recover the DNA fragment, the purified bacteriophage was used astemplate in a PCR reaction with primers flanking the cloning site. A1500-bp DNA fragment was obtained and purified via agarose gelelectrophoresis (10). After EcoR1 digestion, 2 bands of about 800 and700 bp, respectively, appeared on agarose gel (data not shown). Bothfragments were cloned and expressed separately in the SK+ vector. Therecombinant bacteria were then analyzed for the presence of an antigenreactive with the Mab cited above. Only the 800-bp insert produced aprotein which was immunologically reactive with the Mab A66/05H01/E09 asshown by colony blotting. The protein was produced irrespective of IPTGinduction of the culture.

Sequence analysis. By treatment of the 800-bp insert fragment withHindIll, two fragments of 425 bp and 375 bp, respectively, wereobtained. The fragments were cloned in a SK+ vector as EcoR1-HindIIIfragments and sequenced on both strands. The sequence crossing theHindIII site was obtained using internal primers on the parental 800 bpfragment. On one strand, an 463-bp open reading frame (ORF) wasidentified (FIG. 1). The calculated molecular weight of the encodedprotein (17.3 kDa) matched very well with the size observed on Westernblot (FIG. 2). A Shine and Delgarno consensus sequence (GAGGA) was foundupstream from the putative initiation codon (position −8). A consensuspromoter sequence, as described for Brucella genes (12), was not foundin the 290-bp upstream from the ORF which are present in the clonedfragment.

Data bank analysis. A homology search was completed for the 158 aminoacid sequence as shown in FIG. 1 using the Swiss-Prot 27 (all entries)and the PIR 38 (all entries) Data Banks. This homology search on theprotein level confirmed that the 17 kDa Brucella abortus protein asdepicted in SEQ ID NO 2 was new. The closest related proteins found inthis search had a homology of only 36% over the 158 amino acid sequence.Data Bank homology analysis of the nucleotide sequence (SEQ ID NO 1)revealed a homology of only 35% with the closest known sequence (EMBL36;EMBL-nl1011,

Example 2 Production of Recombinant 17-kDa Antigen in E. Coli

2.1. Construction of recombinant vector producing the 17-kDa antigen.

The 800 bp fragment (example 1) was recovered from the SK+ vector by PCRusing two primers designed to allow the directional cloning in theexpression vector pIGFH10. This vector enables expression ofheterologous proteins in E. coli as fusion proteins with a short mousetumor necrosis factor (mTNF) peptide. Moreover, the vector also confersa polyhistidine sequence of six consecutive histidine residues to thefusion protein, allowing fast and efficient purification usingimmobilized metal ion affinity chromatography (IMAC).

The primer used at the 5′-end was 5′CGTGAGGATCCTATGAACCAAAGC3′ (SEQ IDNO 4), whereas the 3′-end primer was 5′GAGTTCTAGACAAGCGCGGCGATGC3′ (SEQID NO 5). The first contains a BamH1 site and the latter an Xba1 site(both in bold) for cloning in the same sites of the expression vectorpIGFH10. In this way a reading frame fusion between the ORF of the 17kDa protein and the mTNF-His6 leader peptide is obtained.

The expected fusion protein will contain 37 amino acids provided by thevector and 158 amino acids from the Brucella gene. The sequence of theexpected protein is given in FIG. 3.

2.2. E. Coli Expression of the 17-kDa Protein.

The transcription of the heterologous gene cloned in the expressionvector pIGFH10 is controlled by the early rightward lamba promoter (Pr)and is controlled by the C1 repressor. When an E. coli strain containinga compatible plasmid with the C1-857 mutant gene encoding a temperaturesensitive variant of the C1 repressor is used, the expression of theheterologous gene can be initiated by shifting the culture from 28° C.to 42° C. For these experiments, the strain MC1061 [pAC1] was used.

This strain was transformed with the expression plasmid and grown at 28°C. For induction experiments, an overnight culture was diluted 1/100 infresh medium containing tetracyclin (10 μg/ml) and grown to a density of0.200 measured at 600 nm.

The temperature was then shifted to 42° C. and incubation was continuedfor several hours. At 1 hour intervals, a sample was taken from theculture for analysis of the expression level. The samples were analysedon PAGE and western blot using Mab A66/05H01/E09.

From these experiments, the conditions for large scale fermentation ofthe culture were determined. A 15L fermentation was then performed usingan induction time of 2 hours. The cells were collected by low speedcentrifugation and the pellet was stored at −70° C. until further use.

2.3. Purification of the 17-kDa Fusion Protein.

The cell pellet was thawed and resuspended in 125 ml of lysis buffer (10mM TrisHCl, 100 mM KCl, 5 mM EDTA, 25 mM aminocaproic acid, 2 mM PMSF, 1mM DTT at pH 6.8) and passed twice through a French press. Aftercentrifugation (27,000 g for 20 min at 4° C.) the pellet was washed with125 ml Triton X-100 buffer (25 mM TrisHCl, 0.05% Triton X-100, pH 6.8)and again centrifuged in the same conditions as above. The pelletobtained was then solubilised in 6M guanidinium chloride buffer (75 ml)(0.1 M Phosphate, 0.05% Triton X-100 at pH 6.5). After clearing at30.000 g for 30 min at 4° C., the solution was loaded on an IMAC columnactivated with NiCl₂ as described by the manufacturer (Pharmacia) andequilibrated with the same 6M guanidinium chloride buffer. The extractwas loaded on the column at room temperature at a flow of 4 ml/min. A 30ml gelbed was used for 37 ml of the extract, containing 18 mg proteinper ml. The column was washed with the 6M guanidinium chloride bufferuntil the absorption measured at 280 nm decreased. Bound proteins werethen eluted with a pH gradient starting at pH 6.5 down to 3.7 (450 ml)and 7 ml fractions were collected.

The eluted proteins were analysed by SDS PAGE and coomassie staining.The 17-kDa protein obtained was estimated to be more than 95% pure andfrom 2 column runs about 350 mg protein was obtained, as estimated bythe BCA method (Pierce) using bovine serum albumine as standard. Theprotein thus obtained was used in competition ELISA assays to study thereaction with Brucella positive sera. Alternatively, this protein wasalso used in indirect ELISA to determine the anti-Brucella antibodiespresent in the sample (see Example 6).

Example 3 Western Blotting of the Recombinant 17-kDa Protein

Western blotting analysis of the protein expressed by the recombinantbacteriophage, containing the above-sequenced region, with MabA66/05H01/E09 showed that the immunoreactive protein migrated as a17-kDa molecule (FIG. 2/A3C3). When Mab A66/05H01/E09 was used todevelop Western blots prepared with lysate from B. melitensis the sameprotein of 17 kDa was revealed (FIG. 2/A3,C3). Another Mab(A68/04G01/C06) known to reveal a 16.5 kDa outer membrane protein(OMP16.5) from B. abortus was also used to probe Western blot stripscontaining the new recombinant 17-kDa antigen as well as anotherrecombinantly produced fusion protein OMP16.5 (Tibor et al., 1994) andB. melitensis extract. This experiment clearly indicates that the lattermonoclonal did not recognize the new 17-kDa antigen, whereas goodreactivity with the recombinant OMP 16.5 was observed as well as withthe Brucella extract (FIG. 2/A2,B2,C2). The D series contains extractfrom E. coli cells, not expressing Brucella proteins and probed with thesame antibodies.

Example 4 Western Blot Analysis of B. melitensis-infected Sheep Sera andB. abortus Infected Cattle Sera

To evaluate the significance of the 17-kDa antigen of the presentinvention for diagnostic purposes, Western blot experiments wereperformed with sera from field-infected animals. These sheep and cattlesera, positive in classical serology, were tested on B. melitensis or B.abortus total protein extract respectively, using Western blot analysis.

From B. melitensis-infected sheep, 100 sera which are positive inclassic standard serology and 20 sera from animals with a negativeserology but a positive DTH reaction, were analyzed by Western blottingon B. melitensis (H38, R strain) total protein extract. Each serumidentified up to 14 bands in the range 10 kDa to 97 kDa (data notshown). In general, more bands with a higher intensity were revealed inthe case of the serologically positive sera than in the case of theserologically negative samples. Out of the 100 and 20 sera mentionedabove, 51% and 20% respectively of these sera showed a band at 17 kDa(Table 2). On the other hand, 36 B. abortus field-infected cow sera wereanalyzed by Western blotting on B. abortus (45/20 R strain) totalprotein extract. Each serum was reactive with up to 12 bands in therange 9 kDa to 97 kDa (data not shown). Thirty-nine percent of these 36sera showed a band at 17 kDa (Table 2). None of the 15 sheep sera northe 14 cow sera from healthy animals gave a signal in the 17 kDa regionupon Western blot analysis.

At the 17-kDa position, a serologically reactive minor outer membraneprotein (OMP16.5) is also present. This protein is distinct from the17-kDa antigen described in the present invention by its reactivitytowards Mabs as well as by its primary sequence (Tibor et al., 1994).

To assess the reaction of the sera with the 17 kDa protein (to ensurethat the reactivity was not due to the OMP16.5), 20 sheep sera and 4 cowsera, randomly chosen from those with a positive serology, and whichreact at 17 kDa in Western blots of Brucella extracts, were retested byWestern blotting on a total protein extract from a recombinant E. coliexpressing the 17-kDa antigen. Seventy percent (14 sera) of the sheepsera and 75% (3 sera) of the cow sera were positive on the recombinantE. coli produced protein (data not shown), indicating that most of thereactivity seen at the 17 kDa position can be attributed to reactionwith the newly identified protein of the invention. In addition, out of10 control sheep sera and 6 control cow sera, none were reactive withthe E. coli produced protein.

From experiments with vaccinated animals, preliminary results indicatethat serological response towards this new 17-kDa antigen may be absentin B19-vaccinated and protected animals whereas it is present in animalsinfected upon challenge. This is an important property which enables theuse of the antigen in differential serology.

Examnple 5 Competition ELISA of Field-infected Sheep and Cow Sera withMab A66/05H01/E09

Fifty sera from field-infected sheep showing reactivity with Brucellaantigens on Western blots, including 23 of which did not react with the17-kDa protein on Western blots, were tested in a competition ELISA withthe Mab A66/05H01/E09. Seventy percent (35 sera) were positive in thistest, including 12 samples which showed no reactivity at the 17 kDaposition on Western blots. Out of the 20 sera from infected sheep with anegative serology but with a positive DTH reaction, 20% could still bedetected by this test. In the same conditions, the 36 sera fromfield-infected cows were tested: sixty-one percent were positive incompetitive ELISA even if only 39% had shown a band at 17 kDa (Table 2).Prom the 10 vaccinated animals, 6 were protected from subsequentinfection, only 3 of which gave healthy calves. The sera from the threelatter heifers did not react in competition ELISA, whereas sera from theother heifers (7 animals) were positive in competition ELISA after theexperimental infection (Table 2). All sera were negative betweenvaccination and infection. The specificity of the reaction was assessedwith 10 sheep sera and 6 cow sera from healthy animals, all of whichwere negative. Furthermore, 2 sera from Yersinia enterocoliticaO:9-infected cows and 2 sera from Salmonella urbana-infected cows werealso negative in the competition ELISA test (data not shown).

Example 6 Indirect ELISA with Recombinantly Produced Brucella 17 kDaFusion Protein.

The purified recombinant fusion protein (Example 2) was used to coatmicrowells (NUNC maxisorp) by diluting the protein to a finalconcentration of 3 μg/ml in phosphate buffered saline pH 6.0. From thissolution, 100 μl was added to each well and incubated for 1 h at 37° C.The wells were then blocked with 0.1% casein solution (blocking buffer)for 1 h at 37° C. and further incubated with 100 μl of the sera to betested, diluted 1/100 in blocking buffer. Bound antibodies were revealedwith a species specific peroxidase conjugated rabbit antibody directedto the Fc part of the IgG. The latter was obtained from DAKO (Denmark)and diluted 1/5000 before use. Substrate and TMB chromophore were thenadded (200 μl) and after 30 min at room temperature the reaction wasstopped with 50 μl 2N sulfuric acid. The absorption was measured at 600nm in an automated ELISA reader.

The results shown in FIG. 4 show that for goat sera of animals infectedwith Brucella, 88% of the animals are reactive with the 17 kDa fusionprotein. For a limited number of human sera tested, a significantfraction also reacted with the 17 kDa protein (FIG. 5). For sheep sera,the fraction reactive with the 17 kDa protein is variable, depending onthe origin of the sera and for cattle, about 45% of infected animals arereactive with the protein. The latter is the highest score ever foundwith a Brucella protein antigen in Brucella field infected cattle.

Table 3 below summarizes the results for the different species tested.

Species Fraction reactive (%) Goats 88 (n = 48) Sheep 37-80 (n > 200)Cattle 45 (n = 35)

Example 6 Construction of Brucella 17-kDa Gene Deletion Mutants forVaccine Purposes

Identification of proteins of interest for diagnosis could be followedby the construction of Brucella vaccinal strains deleted for at leastone of these antigens, assuming that they are essential neither forbacterial survival nor for protection. Diagnosis based on these proteinswould enable the discrimination between vaccinated and infected animals.

6.1. Deletion Strategy

The deletion implies a double homologous recombination event leading tothe replacement of the resident wild type gene ORF (open reading frame)by a genetic marker. Disruption of the gene of interest by insertion ofa genetic marker does not prevent expression of a truncated protein fromthe disrupted gene. This expression may lead to misinterpretation of themutant phenotype. It is therefore preferred to construct mutants with acompletely deleted gene, which can be achieved by an ORF replacementprotocol. The choice of the genetic marker, bioluminescence luxAB gene,will be discussed also.

6.1.1. Choice of a Genetic Marker for Vaccine Strain

The construction of a vaccine strain implies not only a controlledattenuation of the virulence but also the introduction of an appropriategenetic label. Since an antibiotic-resistance marker is an undesirablefeature in a vaccinal strain, the use of a genetic marker like the luxABgene encoding the luciferase from Vibrio harveyi can be envisaged. Theluciferase system is a potentially powerful tool for use in studying thesurvival of genetically engineered bacterial strains in animals and inthe environment. This marker, integrated into the chromosome, has beenused to monitor the survival of engineered Y. enterocolitica in murineand bovine feces (Kaniga et al, 1991).

6.1.2. Principles of the Deletion Strategy.

Reverse genetics, based on homologous recombination, has been greatlyfacilitated by the use of vectors unable to replicate in the host wheremutations are to be introduced. A ColE1-derived plasmid is notmaintained in Brucella species (Halling et al, 1991) and is thereforeuseful as suicide vector.

A ColE1 based suicide vector containing the Brucellia DNA insert wherethe ORF of interest has been replaced by a genetic marker will beintroduced in a recipient Brucelta bacteria by conjugation with an E.coli donor. A mobilizable suicide vector as well as a mobilizing E. colidonor strain are necessary for conjugation to take place. Thesemobilizing E. coli have integrated in their chromosome the transfergenes of the broad host range plasmid RP4 (Simon et al, 1983) and thevector will contain the RK2 origin of transfer (Selvaraj et al, 1984).

By a single homologous recombination between chromosomal and incomingORF flanking regions, plasmid insertion into the chromosomal DNA occurs.This results in an integrant diploid for the flanking regions of thegene of interest. Integrants are obtained by selecting for thevector-borne antibiotic-resistance marker, for example theneomycin-kanamycin phosphotransferase gene encoding kanamycinresistance.

A second homologous recombination event is necessary for plasmidexcision but can result in a wild type genotype, i. e. it leaves a wildtype gene copy in the Brucella chromosome or, on the other hand, canlead to a mutant chromosome. Since the genetic marker used to replacethe ORF (luxAB ) is not a selectable marker, this second event can onlybe detected by screening for the loss of Kan selection marker. Thisprocedure is limiting when the frequency of the second crossing-over islow. To avoid such an efficiency decrease, one can take advantage ofpositive selection of double recombinants using the sacB gene ofBacillus subtilis as described by Kaniga and coll. in 1992 (gene sacB,regulated in cis by the sequence sacR, encodes levan sucrase thatcatalyses hydrolysis of sucrose as well as synthesis of levans).Theseauthors have designed a mobilizable suicide vector containing the sacBselectable marker that allows easy construction of unmarked mutations inGram bacteria species where the expression of sacB gene in presence ofsucrose is lethal. sacB turned out to be an efficient counter-selectionmarker of the double recombination.

Before functional studies are conducted, the deletion mutant has to befurther characterized by southern- and immuno-blotting to ensure thatthe deletion event has taken place as expected.

TABLE 1 Amino acids Synonymous groups Ser (S) Ser, Thr, Gly, Asn Arg (R)Arg, His, Lys, Glu, Gln Leu (L) Leu, Ile, Met, Phe, Val, Tyr Pro (P)Pro, Ala, Thr, Gly Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gln Ala (A)AIa, Pro, Gly, Thr Val (V) Val, Met, Ile, Tyr, Phe, Leu, Val Gly (G)Gly, Ala, Thr, Pro, Ser Ile (I) Ile, Met, Leu, Phe, Val, Tyr Phe (F)Phe, Met, Tyr, Ile, Leu, Trp, Val Tyr (Y) Tyr, Phe, Trp, Met, Ile, Val,Leu Cys (C) Cys, Ser, Thr, Met His (H) His, Gln, Arg, Lys, Glu, Thr Gln(Q) Gln, Glu, His, Lys, Asn, Thr, Arg Asn (N) Asn, Asp, Ser, Gln Lys (K)Lys, Arg, Glu, Gln, His Asp (D) Asp, Asn, Glu, Gln Glu (E) Glu, Gln,Asp, Lys, Asn, His, Arg Met (M) Met, IIe, Leu, Phe, Val

TABLE 2 Sheep sera Field infection Cattle sera Posit. DTH Field inf.Experim. Infect. serol. posit. Healthy posit. serol. P NP HealthyWestern blot # sera tested 100 20 15 36 3 7 6 # posit (%)* 51 (51) 4(20) 0 (0) 14 (39) 0 (0) 5 (71) 0 Competit. ELISA # sera tested 50 20 1036 3 7 6 # positive (%)* 35 (70) 4 (20) 0 (0) 22 (61) 0 (0) 7 (100) 0

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8 1 811 DNA Brucella abortus CDS (290)..(763) 1 gaattccgat cagtgcatagtttccgcgtg ctcgcgcaat ggtgcgcggg cttgttctcg 60 gggcggggtg aaactccccaccggcggtat gaaaagcaat tttcaagccc gcgagcgcct 120 gaaatggaag ccgattcgcatgccatttca gggtcagcag atccggtgag atgccggagc 180 cgacggttaa agtccggatggaagagagcg aatgagcgtc acgattgcgc cttccggcgt 240 cgttcttgcg ttcttttgtgcgccctgatt ctagtttcgt gaggaacct atg aac caa 298 Met Asn Gln 1 agc tgtccg aac aag aca tcc ttt aaa atc gca ttc att cag gcc cgc 346 Ser Cys ProAsn Lys Thr Ser Phe Lys Ile Ala Phe Ile Gln Ala Arg 5 10 15 tgg cac gccgac atc gtt gac gaa gcg cgc aaa agc ttt gtc gcc gaa 394 Trp His Ala AspIle Val Asp Glu Ala Arg Lys Ser Phe Val Ala Glu 20 25 30 35 ctg gcc gcaaag acg ggt ggc agc gtc gag gta gag ata ttc gac gtg 442 Leu Ala Ala LysThr Gly Gly Ser Val Glu Val Glu Ile Phe Asp Val 40 45 50 ccg ggt gca tatgaa att ccc ctt cac gcc aag aca ttg gcc aga acc 490 Pro Gly Ala Tyr GluIle Pro Leu His Ala Lys Thr Leu Ala Arg Thr 55 60 65 ggg cgc tat gca gccatc gtc ggt gcg gcc ttc gtg atc gac ggc ggc 538 Gly Arg Tyr Ala Ala IleVal Gly Ala Ala Phe Val Ile Asp Gly Gly 70 75 80 atc tat cgt cat gat ttcgtg gcg acg gcc gtt atc aac ggc atg atg 586 Ile Tyr Arg His Asp Phe ValAla Thr Ala Val Ile Asn Gly Met Met 85 90 95 cag gtg cag ctt gaa acg gaagtg ccg gtg ctg agc gtc gtg ctg acg 634 Gln Val Gln Leu Glu Thr Glu ValPro Val Leu Ser Val Val Leu Thr 100 105 110 115 ccg cac cat ttc cat gaaagc aag gag cat cac gac ttc ttc cat gct 682 Pro His His Phe His Glu SerLys Glu His His Asp Phe Phe His Ala 120 125 130 cat ttc aag gtg aag ggcgtg gaa gcg gcc cat gcc gcc ttg cag atc 730 His Phe Lys Val Lys Gly ValGlu Ala Ala His Ala Ala Leu Gln Ile 135 140 145 gtg agc gag cgc agc cgcatc gcc gcg ctt gtc tgactaaccc tctataatac 783 Val Ser Glu Arg Ser ArgIle Ala Ala Leu Val 150 155 gcccgcaatg ggtataaatg tcgaattc 811 2 158 PRTBrucella abortus 2 Met Asn Gln Ser Cys Pro Asn Lys Thr Ser Phe Lys IleAla Phe Ile 1 5 10 15 Gln Ala Arg Trp His Ala Asp Ile Val Asp Glu AlaArg Lys Ser Phe 20 25 30 Val Ala Glu Leu Ala Ala Lys Thr Gly Gly Ser ValGlu Val Glu Ile 35 40 45 Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu HisAla Lys Thr Leu 50 55 60 Ala Arg Thr Gly Arg Tyr Ala Ala Ile Val Gly AlaAla Phe Val Ile 65 70 75 80 Asp Gly Gly Ile Tyr Arg His Asp Phe Val AlaThr Ala Val Ile Asn 85 90 95 Gly Met Met Gln Val Gln Leu Glu Thr Glu ValPro Val Leu Ser Val 100 105 110 Val Leu Thr Pro His His Phe His Glu SerLys Glu His His Asp Phe 115 120 125 Phe His Ala His Phe Lys Val Lys GlyVal Glu Ala Ala His Ala Ala 130 135 140 Leu Gln Ile Val Ser Glu Arg SerArg Ile Ala Ala Leu Val 145 150 155 3 195 PRT Artificial SequenceDescription of Artificial SequenceSYNTHETIC CONSTRUCT 3 Met Val Arg SerSer Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His 1 5 10 15 Val Val AlaAsn His Gln Val Glu Glu Gln Gly Ile His His His His 20 25 30 His His ValAsp Pro Met Asn Gln Ser Cys Pro Asn Lys Thr Ser Phe 35 40 45 Lys Ile AlaPhe Ile Gln Ala Arg Trp His Ala Asp Ile Val Asp Glu 50 55 60 Ala Arg LysSer Phe Val Ala Glu Leu Ala Ala Lys Thr Gly Gly Ser 65 70 75 80 Val GluVal Glu Ile Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu 85 90 95 His AlaLys Thr Leu Ala Arg Thr Gly Arg Tyr Ala Ala Ile Val Gly 100 105 110 AlaAla Phe Val Ile Asp Gly Gly Ile Tyr Arg His Asp Phe Val Ala 115 120 125Thr Ala Val Ile Asn Gly Met Met Gln Val Gln Leu Glu Thr Glu Val 130 135140 Pro Val Leu Ser Val Val Leu Thr Pro His His Phe His Glu Ser Lys 145150 155 160 Glu His His Asp Phe Phe His Ala His Phe Lys Val Lys Gly ValGlu 165 170 175 Ala Ala His Ala Ala Leu Gln Ile Val Ser Glu Arg Ser ArgIle Ala 180 185 190 Ala Leu Val 195 4 24 DNA Artificial SequenceDescription of Artificial SequenceSYNTHETIC PRIMER 4 cgtgaggatcctatgaacca aagc 24 5 25 DNA Artificial Sequence Description ofArtificial SequenceSYNTHETIC PRIMER 5 gagttctaga caagcgcggc gatgc 25 611 PRT Brucella abortus 6 Ile Ala Phe Ile Gln Ala Asp Asp Val Leu Lys 15 10 7 9 PRT Brucella abortus VARIANT (7)..(9) Xaa = Any Amino Acid 7Ser Gly Tyr Ile Phe Asp Xaa Pro Gly 1 5 8 14 PRT Brucella abortusVARIANT (6)..(14) Xaa = Any Amino Acid 8 Gly Val Glu Ala Ala Xaa Ala AlaLeu Gln Ile Val Ser Glu 1 5 10

What is claimed is:
 1. An isolated polypeptide wherein the amino acidsequence of the isolated polypeptide has at least 70% identity to the158 residue amino acid sequence as shown in SEQ ID NO
 2. 2. The isolatedpolypeptide of claim 1, wherein the amino acid sequence of the isolatedpolypeptide has at least 80% identity to the 158 residue amino acidsequence as shown in SEQ ID NO
 2. 3. The isolated polypeptide of claim1, wherein the amino acid sequence of the isolated polypeptide has atleast 90% identity to the 158 residue amino acid sequence as shown inSEQ ID NO
 2. 4. An isolated Brucella abortus polypeptide whichcorresponds to an amino acid sequence as shown in SEQ ID NO 2 or SEQ IDNO 3.